CRISP polypeptides as contraceptives and inhibitors of sperm capacitation

ABSTRACT

Included in the present invention are methods of inhibiting sperm capacitation, inhibiting the phosphorylation of a protein at tyrosine residues, inhibiting an acrosomal reaction, and inhibiting fertilization of an egg by sperm with the administration of a CRISP polypeptide.

STATEMENT OF GOVERNMENT RIGHTS

This invention was made with government support under a grant from theNational Institutes of Health, Grant No. HD 11962. The U.S. Governmenthas certain rights in this invention.

This application is a national stage filing of International PatentApplication No. PCT/US03/16669, filed May 28, 2003, which in turn claimspriority to U.S. Provisional Application Ser. No. 60/383,628, filed May28, 2002, both of which are incorporated herein by reference in theirentirety.

BACKGROUND

An effective, safe and easily reversible male contraceptive withuniversal acceptability remains an elusive goal. Although a variety ofapproaches for achieving male contraception have been tried, no singlemode of male contraception is without its immediate drawbacks forefficacy or compliance. Even seemingly simple interventions have notproven to be widely acceptable. For example, surgical or non-surgicalvasectomy, methods that interrupt sperm transport in the malereproductive tract, are not without their complications or long-termrisk. More complex approaches, such as regimens for the hormonal controlof male fertility, have also not been fully satisfactory. Such methodshave focused on the suppression of spermatogenesis to the point ofazoospermia, a goal that has been difficult to achieve. The use of theimmune response to block contraception has been an important front inefforts to develop more sophisticated contraceptive systems.Unfortunately, such approaches have thus far failed, as maleautoimmunity against sperm does not suppress sperm production in men;this is known because such autoimmunity can occur after vasectomy. Thus,inhibiting sperm fertilizing-ability without affecting the hormonalbalance in either the male or female remains an important goal in thefield of reproductive biology. The present invention achieves this goal.

SUMMARY OF THE INVENTION

The present invention includes a method of inhibiting sperm capacitationincluding contacting sperm with a CRISP polypeptide. Also included inthe present invention is a method of inhibiting sperm capacitation in anindividual including the administration of a CRISP polypeptide to theindividual.

In another aspect, the present invention also includes a method forinhibiting fertilization of an egg by sperm in an individual, comprisingthe administration of a CRISP polypeptide to the individual.

In another aspect, the present invention includes a method of inhibitingthe phosphorylation of a protein at tyrosine residues includingcontacting the protein with a CRISP polypeptide. In some embodiments ofthe present invention, the protein may be on the surface of mammaliansperm.

A further aspect of the present invention includes a method ofinhibiting an acrosomal reaction including contacting the acrosomalreaction with a CRISP polypeptide.

In some embodiments of the methods of the present invention, the CRISPpolypeptide may be administered orally. In some embodiments of themethods of the present invention, the CRISP polypeptide may beadministered parenterally. In some embodiments of the methods of thepresent invention, the CRISP polypeptide may be administeredtransdermally. In some embodiments of the methods of the presentinvention, the CRISP polypeptide may be administered in a compositionincluding a pharmaceutically acceptable carrier.

In some embodiments of the methods of the present invention, theindividual may be a mammalian male. In some embodiments of the methodsof the present invention, the individual may be a mammalian female. Insome embodiments of the methods of the present invention, the CRISPpolypeptide may be administered intravaginally, including administeredas a time released, vaginal implant. In other embodiments of the methodsof the present invention, the CRISP polypeptide is administered to thevagina of the mammalian female in an amount capable of inhibiting spermcapacitation, rendering said sperm incapable of fertilization.

In other embodiments of the methods of the present invention, the CRISPpolypeptide has at least about 40% structural identity to a polypeptideselected from the group consisting of human CRISP-1 (SEQ ID NO:1, ratCRISP-1 (SEQ ID NO:2), mouse CRISP-1 (SEQ ID NO:3), human CRISP-2 (SEQID NO:4), rat CRISP-2 (SEQ ID NO:5), human CRISP-3 (SEQ ID NO:6), mouseCRISP-3 (SEQ ID NO:7), and biologically active analogs thereof.

In yet other embodiments of the methods of the present invention, theCRISP polypeptide has at least about 40% structural identity to humanCRISP-1 (SEQ ID NO:1) or a biologically active analog thereof. In someembodiments of the methods of the present invention, the CRISPpolypeptide is human CRISP-1 (SEQ ID NO:1).

In other embodiments of the methods of the present invention, the CRISPpolypeptide has about at least 40% structural identity to rat CRISP-1(SEQ ID NO:2) of a biologically active analog thereof. In someembodiments of the methods of the present invention, the CRISPpolypeptide is rat CRISP-1 (SEQ ID NO:2).

Also included in the present invention is a contraceptive compositionincluding a CRISP polypeptide in an amount effective to inhibit spermcapacitation, inhibit phosphorylation of a protein at tyrosine residues,inhibit an acrosome reaction, and/or inhibit fertilization of an egg bysperm. In some embodiments of the contraceptive composition of thepresent invention, the CRISP polypeptide has at least about 40%structural identity to a polypeptide selected from the group consistingof human CRISP-1 (SEQ ID NO:1, rat CRISP-1 (SEQ ID NO:2), mouse CRISP-1(SEQ ID NO:3), human CRISP-2 (SEQ ID NO:4), rat CRISP-2 (SEQ ID NO:5),human CRISP-3 (SEQ ID NO:6), mouse CRISP-3 (SEQ ID NO:7) andbiologically active analogs thereof. In some embodiments of thecontraceptive composition of the present invention, the CRISPpolypeptide has at least about 40% structural identity to human CRISP-1(SEQ ID NO:1) and biologically active analogs thereof. In someembodiments of the contraceptive composition of the present invention,the CRISP polypeptide is human CRISP-1 (SEQ ID NO:1). In someembodiments of the contraceptive composition of the present invention,the CRISP polypeptide has at least about 40% structural identity to ratCRISP-1 (SEQ ID NO:2), and biologically active analogs thereof. In otherembodiments of the contraceptive composition of the present invention,the CRISP polypeptide is rat CRISP-1 (SEQ ID NO:2). In some embodimentsof the contraceptive composition of the present invention, thecontraceptive composition further includes a spermicidal or an antiviralagent.

DETAILED DESCRIPTION OF THE DRAWINGS

FIG. 1. Immunoblot of solubilized rat sperm collected from the end ofthe epididymis and incubated in a defined capacitation medium in vitrofor 5 hours under controlled conditions. A sample of sperm was taken atthe beginning of incubation to provide the time zero conditions (lane1). Aliquots of collected sperm were incubated under the followingvarious conditions: 5 hours under non-capacitation conditions (lane 2);5 hours under capacitating conditions (lane 3); and 5 hours undercapacitating conditions with increasing concentrations of CRISP-1 (lanes4, 5, and 6). In FIG. 1A, the immunoblot is stained with ananti-phosphotyrosine antibody. FIG. 1B shows the same gel stained withan anti-CRISP-1 antibody.

FIG. 2. The requirement of incubating rat sperm with bovine serumalbumin (BSA) to achieve the tyrosine phosphorylation associated withcapacitation. Rat epididymal sperm were isolated in BWW with (+BSA) orwithout (−BSA) 15 mg/ml lipid-rich BSA for 5 hours. Aliquots of spermwere checked for tyrosine phosphorylation at 1, 3, and 5 hour timepoints by western blot analysis using an anti-phosphotyrosine antibody(FIG. 2A). A steady accumulation of tyrosine phosphorylation wasobserved in the presence of BSA, with negligible phosphorylation in BWWalone. To determine if extraction of cholesterol was the action of theBSA that led to the tyrosine phosphorylation, sperm were incubated with15 mg/ml BSA with (Ch) or without (B) the addition of 30 μM cholesterolsulfate and tyrosine phosphorylation compared to levels seen in spermincubated in BWW alone (C). The addition of exognenous cholesterolsulfate eliminated the BSA-induced phosphorylation of sperm proteins(FIG. 2B).

FIG. 3. The comparative activity of lipid-rich BSA and Fraction V BSA inthe induction of protein tyrosine phosphorylation of rat and mousesperm. Rat epididymal sperm were incubated in increasing concentrationsof lipid-rich (LR) or Fraction V (F5) BSA and protein tyrosinephosphorylation was determined after 5 hours (FIG. 3A). Both lipid-richand Fraction V BSA showed maximal induction of phosphorylation at 15mg/ml, but phosphorylation was greatest in lipid-rich BSA at eachconcentration. Since Fraction V BSA is routinely used for capacitationstudies in other species, 4 mg/ml lipid-rich or Fraction V BSA weretested in capacitation incubations with mouse sperm (FIG. 3B). Withmouse sperm, both lipid-rich and Fraction V are equipotent at inducingprotein tyrosine phosphorylation.

FIG. 4. The requirement of extracellular Ca⁺⁺ for induction ofcapacitation in rat sperm. Sperm were incubated in BWW solution with(+Ca⁺⁺) or without (−Ca⁺⁺) 1.7 mM Ca⁺⁺. At hourly time points out to 4hours, sperm were tested for the presence of tyrosine-phosphorylatedproteins. The presence of extracellular calcium ion was required formaximal phosphorylation.

FIG. 5. The requirement of extracellular bicarbonate ion for inductionof capacitation in rat sperm. Sperm were incubated in BWW solution with(+) or without (−) 25 mM HCO₃ ⁻ for 4 hours and then tested for thepresence of tyrosine-phosphorylated proteins. The presence ofbicarbonate ion in the media was required for tyrosine phosphorylationof sperm proteins. Omission of bicarbonate resulted in phosphorylationlevels the same as BWW alone (C).

FIG. 6. Quantitative kinetics of cholesterol extraction and proteintyrosine phosphorylation of sperm proteins. Rat epididymal sperm wereincubated in 1 or 2 mM methyl-β-cyclodextran (MBCD) and extractedcholesterol measured at time intervals out to 2 hours (FIG. 6A). Thelevels of cholesterol were determined by the Amplex Red Cholesterolassay and the results normalized to cholesterol extracted in BWW alone.Protein tyrosine phosphorylation was measured by western blot at hourlytime points during extraction with MBCD (FIG. 6B). Cholesterolextraction reached a plateau with 1 mM MBCD at 30 minutes and with 2 mMMBCD between 60 and 120 minutes (later time points not shown). Maximalphosphorylation lagged behind maximal cholesterol extraction with bothconcentrations of MBCD.

FIG. 7. The effect of incubation of rat epididymal sperm with exogenouspurified Crisp-1 on the level of protein tyrosine phosphoryation. Spermwere incubated under capacitating conditions with 15 mg/ml lipid-richBSA for 5 hours in the presence of increasing concentrations (μg/ml) ofpurified proteins DE (FIG. 7A). Analysis of cells prior to capacitationincubation are shown as control (C). At 400 μg/ml protein tyrosinephosphorylation was nearly completely inhibited. The same Western blotwas stripped and probed with antibody CAP-A (FIG. 7B) and 4E9 (FIG. 7C).Protein detected by CAP-A demonstrates that Crisp-1 re-associates withthe sperm in a dose dependent fashion that correlates with theinhibition of capacitation (FIG. 7B). Antibody CAP-A detects all formsof Crisp-1 including processed forms of proteins D and E. Monoclonalantibody 4E9 detects only forms of Protein E (FIG. 7C). Comparison ofthe staining with 4E9, which stains only a processed form of protein Eextracted from the sperm surface, and CAP-A demonstrates that only anunprocessed form of protein D re-associates with sperm to inhibitphosphorylation. The unprocessed Crisp-1 detected by CAP-A is lost withtime when the sperm are removed from the exogenous pure Crisp-1solution, suggesting that unprocessed Crisp-1 associates in areceptor-ligand fashion while processed Crisp-1 is covalently attachedto the sperm surface.

FIG. 8. The effect of incubation of rat epididymal sperm with exogenouspurified Crisp-1 on the level of progesterone induced acrosome reaction.Sperm were incubated under capacitating conditions for 1 hours in thepresence or absence of 400 μg/ml Crisp-1. Progesterone (P4) at 1 μM wasadded to sperm after 30 minutes of incubation to induce the acrosomereaction. DMSO, the solvent used for the P4 stock solution, was added tocontrol cells. Addition of P4 to capacitated sperm (+BSA+P4) caused astatistically significant (*P<0.05) increase in acrosome reacted spermcompared to capacitated sperm (+BSA or +BSA+DMSO). This increase iscompletely abolished by addition of exogenous Crisp-1 (+BSA+P4+CRISP-1),as evidenced by the statistically significant decrease (*P<0.05) inacrosome reacted sperm. Columns are shown with values at base. Thepercent acrosome reacted sperm in the +BSA+P4 group was significantlyhigher (*P<0.05) than all other groups and there was no significantdifference in the percent acrosome reacted sperm between any of theother groups. Data are presented as means +/−SEM.

FIG. 9. The reversibility of protein tyrosine phosphoryation inhibitionby exogenous purified Crisp-1 in rat epididymal sperm. Sperm wereincubated under capacitating conditions with 15 mg/ml lipid-rich BSA for5 hours in the presence (lane 4) or absence (lane 3) of 200 μg/mlCrisp-1. At 5 hours sperm were washed free of exogenous Crisp-1 andincubated for an additional 3 (lane 5) or 19 (lane 6) hours. Sperm attime zero and after 5 hours in BWW without BSA are shown in lanes 1 and2, respectively. Sperm proteins were analyzed by western blot analysisfor protein tyrosine phosphorylation (FIG. 9A) and Crisp-1 (FIG. 9B). Asshown previously, 200 μg/ml Crisp-1 has an inhibitory effect on spermprotein tyrosine phosphorylation. The inhibition of protein tyrosinephosphorylation was reversed with the removal of Crisp-1. ExogenousCrisp-1 associated with sperm after 5 hours incubation is lost from thesurface of sperm with time. An aliquot of purified Crisp-1 used in thesperm incubations is shown in lane 7.

FIG. 10. Effect of exogenous administration of cAMP analog, dibromo-cAMP(db-cAMP), and the phosphodiesterase inhibitor IBMX on the proteintyrosine phosphorylation associated with capacitation. To determine ifBSA, Ca⁺⁺, and HCO₃ ⁻ act upstream of cAMP in the signaling cascade thatleads to protein tyrosine phosphorylation (FIG. 10A), sperm wereincubated in the presence (lanes 4, 6, 8) or absence (lanes 3, 5, 7) ofdb-cAMP/IBMX without BSA (lanes 3, 4), Ca⁺⁺ (lanes 5, 6), or HCO₃ ⁻(lanes 7, 8). Control phosphorylation in BWW or BWW with BSA are shownin lanes 1 and 2, respectively. In each case, exogenous cAMP and IBMXovercome the block to phosphorylation caused by omission of BSA, Ca⁺⁺,or HCO₃ ⁻ from the capacitation medium, indicating that cAMP actsdownstream for the effect of these three required constituents ofcapacitation. The ability of cAMP to overcome the inhibition ofphosphorylation by Crisp-1 was tested by incubating sperm undercapacitating conditions with and without db-cAMP/IBMX in the presence of400 μg/ml pure Crisp-1 (lanes 3 & 4, respectively, FIG. 10B). Controlsperm in BWW only or BWW with BSA are shown in lanes 1 and 2,respectively. The results show that the block to phosphorylation causedby Crisp-1 is also upstream of the effect of cAMP on protein tyrosinephosphorylation.

FIG. 11. Amino acid sequence of human CRISP-1 (SEQ ID NO:1), rat CRISP-1(SEQ ID NO:2), mouse CRISP-1 (SEQ ID NO:3), human CRISP-2 (SEQ ID NO:4),rat CRISP-2 (SEQ ID NO:5), human CRISP-3 (SEQ ID NO:6) and mouse CRISP-3(SEQ ID NO7).

FIG. 12. cDNA sequences encoding human CRISP-1 (SEQ ID NO:8), ratCRISP-1 (SEQ ID NO:9), mouse CRISP-1 (SEQ ID NO:10), human CRISP-2 (SEQID NO:11), rat CRISP-2 (SEQ ID NO:12), human CRISP-3 (SEQ ID NO:13) andmouse CRISP-3 (SEQ ID NO:14).

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

With the present invention it has been demonstrated that a CRISPpolypeptide inhibits sperm capacitation, inhibits proteinphosphorylation at tyrosine residues, and inhibits the acrosomalreaction. Thus, CRISP polypeptides can be used in improved methods ofcontraception, without affecting or interfering with the hormonal orimmune systems. The CRISP polypeptides of the present invention includenaturally occurring CRISP polypeptides and biologically active analogsthereof.

Naturally occurring CRISP polypeptides comprise a family ofCysteine-RIch Secretory Proteins that are expressed in numerous organsin male animals, particularly in the reproductive tract. CRISPpolypeptides are not generally expressed in female animals, with theexception of neutrophils, and possibly in tumors. In the male, CRISP-1is expressed primarily in the epididymis, CRISP-2 is expressed primarilyin the testis and CRISP-3 is expressed primarily in salivary glands.Prostate and seminal vesicles also have low expression of some of theseproteins. Sperm require passage through the epididymis before they areable to fertilize an egg. This passage is an obligatory maturationalprocess in the male and during this time, CRISP-1, a secretory productof the epididymis, is added to the sperm surface. When sperm areejaculated into the female reproductive tract they under go a processcalled “capacitation,” which is required as the final maturational stepbefore interaction between sperm and egg. It is well recognized thatsperm that are not capacitated win not fertilize. Thus, theidentification of agents that inhibit capacitation will lead todevelopment of improved contraceptives.

The CRISP family of polypeptides has been extensively characterized andthe amino acid sequences of the CRISP-1, CRISP-2 and CRISP-3polypeptides from a number of species are known. The CRISP-1polypeptides from human (Kratzschmar et al., Eur. J. Biochem.236(3):827-36, 1996), rat (Klemme et al., Gene 240(2):279-88, 1999;Charest et al., Mol. Endocrinol. 2 (10), 999-1004, 1988; Brooks et al.,Eur. J. Biochem 161(1):13-18, 1986), and mouse (Eberspaecher et al.,Mol. Reprod. Dev. 42:157-172, 1995; Haendler et al., Endocrinology 133(1), 192-198, 1993) have been characterized. The human CRISP-1 aminoacid sequence (SEQ ID NO:1) is available as Genbank Accession NumberCAA64524, the rat CRISP-1 amino acid sequence (SEQ ID NO:2) is availableas Genbank Accession Number AAD41529, and the mouse CRISP-1 amino acidsequence (SEQ ID NO:3) is available as Genbank Accession A49202, all ofwhich are shown in FIG. 11. The cDNA sequence encoding human CRISP-1(SEQ ID NO:8) is available as Genbank Accession Number X95237, the cDNAsequence encoding rat CRISP-1 (SEQ ID NO:9) is available as GenbankAccession Number NM_(—)022859, and the cDNA sequence encoding mouseCRISP-1 (SEQ ID NO:10) is available as Genbank Accession Number L05559,all of which are shown in FIG. 12.

The CRISP-2 polypeptides from human (Kratzschmar et al., Eur. J.Biochem. 236 (3), 827-836, 1996) and rat (O'Bryan et al., Mol. Reprod.Dev. 50 (3), 313-322, 1998) have been characterized. The human CRISP-2amino acid sequence (SEQ ID NO:4), available as Genbank Accession NumberP16562, and the rat CRISP-2 amino acid sequence (SEQ ID NO:5), availableas Genbank Accession Number AAD48090, are shown in FIG. 11. The cDNAsequence encoding human CRISP-2 (SEQ ID NO:11) is available as GenbankAccession Number X95239 and the cDNA sequence encoding rat CRISP-2 (SEQID NO:12) is available as Genbank Accession Number AF078552, all ofwhich are shown in FIG. 12.

The CRISP-3 polypeptides from human (Kratzschmar et al., Eur. J.Biochem. 236 (3), 827-836, 1996) and mouse (Haendler et al.,Endocrinology 133 (1), 192-198 (1993)) have been characterized. Thehuman CRISP-3 amino acid sequence (SEQ ID NO:6), available as GenbankAccession Number P54108, and the mouse CRISP-3 amino acid sequence (SEQID NO:7), available as Genbank Accession Number Q03402, are shown inFIG. 11. The cDNA sequence encoding human CRISP-3 (SEQ ID NO:13) isavailable as Genbank Accession Number X95240 and the cDNA sequenceencoding mouse CRISP-3 (SEQ ID NO:14) is available as Genbank AccessionNumber L05560, all of which are shown in FIG. 12.

The CRISP polypeptides of the present invention may be derived from avariety of species, including, but not limited to, human, primate, rat,mouse, bovine, and horse. The CRISP polypeptides of the presentinvention include, but are not limited to, CRISP-1, CRISP-2 and CRISP-3polypeptides. For example, the CRISP polypeptides of the presentinvention include, but are not limited to, human CRISP-1 (SEQ ID NO:1),rat CRISP-1 (SEQ ID NO:2), mouse CRISP-1 (SEQ ID NO:3), human CRISP-2(SEQ ID NO:4), rat CRISP-2 (SEQ ID NO:5), human CRISP-3 (SEQ ID NO:6),and mouse CRISP-3 (SEQ ID NO:7).

“Polypeptide” as used herein refers to a polymer of amino acids and doesnot refer to a specific length of a polymer of amino acids. Thus, forexample, the terms peptide, oligopeptide, protein, and enzyme areincluded within the definition of polypeptide, whether naturallyoccurring or synthetically derived, for instance, by recombinanttechniques or chemically or enzymatically synthesized. This term alsoincludes post-expression modifications of the polypeptide, for example,glycosylations, acetylations, phosphorylations, and the like. Thefollowing abbreviations are used throughout the application:

-   A=Ala=Alanine-   V=Val=Valine-   L=Leu=Leucine-   I=Ile=Isoleucine-   P=Pro=Proline-   F=Phe=Phenylalanine-   W=Trp=Tryptophan-   M=Met=Methionine-   G=Gly=Glycine-   S=Ser=Serine-   T=Thr=Threonine-   C=Cys=Cysteine-   Y=Tyr=Tyrosine-   N=Asn=Asparagine-   Q=Gln=Glutamine-   D=Asp=Aspartic Acid-   E=Glu=Glutamic Acid-   K=Lys=Lysine-   R=Arg=Arginine-   H=His=Histidine

As used herein, a CRISP polypeptide also includes “biologically activeanalogs” of naturally occurring CRISP polypeptides. For example, theCRISP polypeptides of the present invention include, but are not limitedto, biologically active analogs of human CRISP-1 (SEQ ID NO:1), ratCRISP-1 (SEQ ID NO:2), mouse CRISP-1 (SEQ ID NO:3), human CRISP-2 (SEQID NO:4), rat CRISP-2 (SEQ ID NO:5), human CRISP-3 (SEQ ID NO:6), ormouse CRISP-3 (SEQ ID NO:7).

As used herein to describe a CRISP polypeptide, the term “biologicallyactive” means to inhibit protein tyrosine phosphorylation, inhibit spermcapacitation, inhibit an acrosome reaction, and/or inhibit fertilizationof an egg by sperm. Biological activity of a CRISP polypeptide can beeasily assessed using the various assays described herein as well asother assays well known to one with ordinary skill in the art. Aninhibition in biological activity can be readily ascertained by thevarious assays described herein, and by assays known to one of skill inthe art. An inhibition in biological activity can be quantitativelymeasured and described as a percentage of the biological activity of acomparable control. The biological activity of the present inventionincludes an inhibition that is at least about 5%, at least about 10%, atleast about 15%, at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 55%, at least about 60%, at least about65%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about99%, at least about 100%, at least about 110%, at least about 125%, atleast about 150%, at least about 200%, at least or about 250% of theactivity of a suitable control.

A “biologically active analog” of a CRISP polypeptide includespolypeptides having one or more amino acid substitutions that do noteliminate biological activity. Substitutes for an amino acid in thepolypeptides of the invention may be selected from other members of theclass to which the amino acid belongs. For example, it is well-known inthe art of protein biochemistry that an amino acid belonging to agrouping of amino acids having a particular size or characteristic (suchas charge, hydrophobicity and hydrophilicity) can be substituted foranother amino acid without altering the activity of a protein,particularly in regions of the protein that are not directly associatedwith biological activity. Substitutes for an amino acid may be selectedfrom other members of the class to which the amino acid belongs. Forexample, nonpolar (hydrophobic) amino acids include alanine, leucine,isoleucine, valine, proline, phenylalanine, tryptophan, and tyrosine.Polar neutral amino acids include glycine, serine, threonine, cysteine,tyrosine, asparagine and glutamine. The positively charged (basic) aminoacids include arginine, lysine and histidine. The negatively charged(acidic) amino acids include aspartic acid and glutamic acid. Examplesof such preferred conservative substitutions include Lys for Arg andvice versa to maintain a positive charge; Glu for Asp and vice versa tomaintain a negative charge; Ser for Thr so that a free —OH ismaintained; and Gln for Asn to maintain a free NH2. Likewise,biologically active analogs of a CRISP polypeptide containing deletionsor additions of one or more contiguous or noncontiguous amino acids thatdo not eliminate the biological activity of the CRISP polypeptide arealso contemplated.

A “biologically active analog” of a CRISP polypeptide includes“fragments” and “modifications” of a CRISP polypeptide. As used herein,a “fragment” of a CRISP polypeptide means a CRISP polypeptide that hasbeen truncated at the N-terminus, the C-terminus, or both. The CRISPprotein family is characterized by sixteen-conserved cysteine residueslocated within the C-terminus of the polypeptide. A “fragment” of aCRISP polypeptide may include 1, 2, 3, 4, 5 6, 7, 8, 9, 10, 11, 12, 13,14, 15, or 16 of the conserved cysteine residues of the CRISP proteinfamily. A fragment may range for about 5 to about 250 amino acids inlength. For example it may be about 5, about 10, about 20, about 25,about 50, about 75, about 100, about 125, about 150, about 175, about200, about 225, or about 250 amino acids in length. Fragments of a CRISPpolypeptide with potential biological activity can be identified by manymeans. One means of identifying such fragments of a CRISP polypeptidewith biological activity is to compare the amino acid sequences of aCRISP polypeptide from rat, mouse, human and/or other species to oneanother. Regions of homology can then be prepared as fragments.

A “modification” of a CRISP polypeptide includes CRISP polypeptides orfragments thereof chemically or enzymatically derivatized at one or moreconstituent amino acid, including side chain modifications, backbonemodifications, and N- and C-terminal modifications includingacetylation, hydroxylation, methylation, amidation, and the attachmentof carbohydrate or lipid moieties, cofactors, and the like. Modifiedpolypeptides of the invention may retain the biological activity of theunmodified polypeptide or may exhibit a reduced or increased biologicalactivity.

The CRISP polypeptides and biologically active analogs thereof of thepresent invention include native (naturally occurring), recombinant, andchemically or enzymatically synthesized polypeptides. For example, theCRISP polypeptides of the present invention may be prepared followingthe procedures for the isolation of CRISP-1 polypeptide from rat spermdetailed by Hall and Tubbs (Prep. Biochem. Biotechnol. 27(4):239-51,1997). For example, the CRISP polypeptides of the present invention canbe prepared recombinantly, by well known methods, including, forexample, preparation as fusion proteins in bacteria and insect cells.

As used herein, the term “isolated” means that a polynucleotide orpolypeptide is either removed from its natural environment orsynthetically derived, for instance by recombinant techniques, orchemically or enzymatically synthesized. An isolated polynucleotidedenotes a polynucleotide that has been removed from its natural geneticmilieu and is thus free of other extraneous or unwanted codingsequences, and is in a form suitable for use within geneticallyengineered protein production systems. Isolated polynucleotides of thepresent invention are free of other coding sequences with which they areordinarily associated, but may include naturally occurring 5′ and 3′untranslated regions such as promoters and terminators. Preferably, thepolynucleotide or polypeptide is purified, i.e., essentially free fromany other polynucleotides or polypeptides and associated cellularproducts or other impurities.

As used herein, “structural similarity” refers to the identity betweentwo polypeptides. Structural similarity is generally determined byaligning the residues of the two polypeptides to optimize the number ofidentical amino acids along the lengths of their sequences; gaps ineither or both sequences are permitted in making the alignment in orderto optimize the number of identical amino acids, although the aminoacids in each sequence must nonetheless remain in their proper order.For example, polypeptides may be compared using the Blastp program ofthe BLAST 2 search algorithm, as described by Tatusova et al. (FEMSMicrobiol. Lett., 174; 247-250, 1999) and available on the world wideweb at ncbi.nlm.nih.gov/BLAST/. The default values for all BLAST 2search parameters may be used, including matrix=BLOSUM62; open gappenalty=11, extension gap penalty=1, gap x_dropoff=50, expect=10,wordsize=3, and filter on. In the comparison of two amino acid sequencesusing the BLAST search algorithm, structural similarity may be referredto by percent “identity” or may be referred to by percent “similarity.”“Identity” refers to the presence of identical amino acids and“similarity” refers to the presence of not only identical amino acidsbut also the presence of conservative substitutions.

The CRISP polypeptides of the present invention include polypeptideswith at least about 40%, at least about 45%, at least about 50%, atleast about 55%, at least about 60%, at least about 65%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, or at least about 99% structuralidentity to a known rat, mouse or human CRISP polypeptide. The CRISPpolypeptides of the present invention also include polypeptides with atleast about 40%, at least about 45%, at least about 50%, at least about55%, at least about 60%, at least about 65%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, or at least about 99% structural similarity toa known rat, mouse or human CRISP polypeptide.

For example, the CRISP polypeptides of the present invention mayinclude, but are not limited to, polypeptides with at least about 40%,at least about 45%, at least about 50%, at least about 55%, at leastabout 60%, at least about 65%, at least about 70%, at least about 75%,at least about 80%, at least about 85%, at least about 90%, at leastabout 95%, or at least about 99% structural identity to human CRISP-1(SEQ ID NO:1), rat CRISP-1 (SEQ ID NO:2), or mouse CRISP-1 (SEQ IDNO:3). For example, the CRISP polypeptides of the present invention mayalso include, but are not limited to, polypeptides with at least about40%, at least about 45%, at least about 50%, at least about 55%, atleast about 60%, at least about 65%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, or at least about 99% structural similarity to humanCRISP-1 (SEQ ID NO:1), rat CRISP-1 (SEQ ID NO:2), or mouse CRISP-1 (SEQID NO:3).

According to the present invention, a CRISP polypeptide, includingbiologically active analogs thereof, can be administered to a subject inan effective amount sufficient to inhibit protein phosphorylation attyrosine residues, inhibit sperm capacitation, inhibit an acrosomereaction, and/or inhibit the fertilization of an egg by sperm. The CRISPpolypeptides of the present invention may be administered to a male orfemale individual. The individual may be a mammal, including, but notlimited to a mouse, rat, primate, bovine, or human. For example, in oneembodiment of the present invention, a CRISP-1 polypeptide, or abiologically active analog thereof, can be administered to a subject inan effective amount sufficient to inhibit protein phosphorylation attyrosine residues, inhibit sperm capacitation, inhibit an acrosomereaction, and/or inhibit the fertilization of an egg by sperm.

As used herein an “acrosome reaction” or “acrosomal reaction” includesthe sequence of structural changes that occur in spermatozoa when in thevicinity of an oocyte. Such structural changes serve to facilitate entryof a spermatozoon into the oocyte and include the fusion of portions ofthe outer membrane of the acrosome with the plasma membrane of the spermhead, creating openings through which the enzymes of the acrosome arereleased. See, for example, Wasserman et al., Nat. Cell Biol. 3:9-14,2001.

By the term “effective amount” of an agent as provided herein is meant anontoxic but sufficient amount of the agent or composition to providethe desired effect. Thus, in the context of the present invention, an“effective amount” of a CRISP polypeptide is an amount sufficient toinhibit protein phosphorylation at a tyrosine residue, inhibit spermcapacitation, inhibit an acrosome reaction, and/or affect contraception.The exact amount required will vary from subject to subject, dependingon the species, age, and general condition of the subject, the severityof the condition being treated, and the particular agent and its mode ofadministration, and the like. Thus, it is not possible to specify anexact “effective amount.” However, an appropriate effective amount maybe determined by one of ordinary skill in the art using only routineexperimentation. Therapeutically effective concentrations and amountsmay be determined for each application herein empirically by testing thecompounds in known in vitro and in vivo systems, such as those describedherein; dosages for humans or other animals may then be extrapolatedtherefrom.

In some embodiments of the present invention, a CRISP polypeptide may bedelivered by intravaginal administration. For such administration, aCRISP polypeptide may be provided as a cream gel, foam, emulsion,suppository, and the like. In certain embodiments of the presentinvention, CRISP polypeptides may be contained within a time releasedvaginal implant.

In some embodiments of the present invention, a CRISP polypeptide may bedelivered by oral administration. For such oral administration, a CRISPpolypeptide may be provided as a liquid, a tablet, a pill, a capsule, agel coated tablet, a syrup, or some other oral administration method. Incertain embodiments of the present invention, CRISP polypeptides may becontained within a bio-erodible matrix for time-controlled release.

In some embodiments of the present invention, a CRISP polypeptide may bedelivered by transdermal administration. For such administration, aCRISP polypeptide may be provided as a cream, a transdermal patch, andthe like. In certain embodiments of the present invention, CRISPpolypeptides may be contained within a time released composition.

In some embodiments of the present invention, a CRISP polypeptide may bedelivered by parenteral administration. For such administration, a CRISPpolypeptide may by provided in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous, intraperitoneal, and intratumoraladministration. In this connection, sterile aqueous media that can beemployed will be known to those of skill in the art in light of thepresent disclosure (see for example, “Remington's PharmaceuticalSciences” 15th Edition). Some variation in dosage will necessarily occurdepending on the condition of the subject being treated. The personresponsible for administration will, in any event, determine theappropriate dose for the individual subject. Moreover, for humanadministration, preparations should meet sterility, pyrogenicity, andgeneral safety and purity standards as required by the FDA.

In some aspects, the present invention includes contraceptivecompositions including an effective amount of a CRISP polypeptide, or abiologically active analog thereof, in an amount effective to inhibitsperm capacitation, inhibit protein tyrosine phosphorylation, inhibit anacrosome reaction, and/or effect contraception. These contraceptivecompositions may contain one or more active agents. For example, suchcontraceptive compositions may include, one or more CRISP polypeptides.Such contraceptive compositions may include one or more additionalactive agents that are not a CRISP polypeptide. Such active agents mayinclude, but are not limited to, spermicidal agents and/or antiviralagents.

The CRISP polypeptides of the present invention may be administered atonce, or may be divided into a number of smaller doses to beadministered at intervals of time. It is understood that the precisedosage and duration of treatment is a function of the desiredtherapeutic outcome and may be determined empirically using knowntesting protocols or by extrapolation from in vivo or in vitro testdata. It is to be noted that concentrations and dosage values may alsovary with the severity of the condition to be treated. It is to befurther understood that for any particular subject, specific dosageregimens should be adjusted over time according to the individual needand the professional judgment of the person administering or supervisingthe administration of the compositions, and that the concentrationranges set forth herein are exemplary only and are not intended to limitthe scope or practice of the claimed compositions and methods. Theagents of the present invention may be administered to the subject incombination with other modes of contraception. The agents of the presentinvention can be administered before, during or after the administrationof the other therapies.

The CRISP polypeptides of the present invention may be formulated in acomposition along with a “carrier.” As used herein, “carrier” includesany and all solvents, dispersion media, vehicles, coatings, diluents,antibacterial and antifungal agents, isotonic and absorption delayingagents, buffers, carrier solutions, suspensions, colloids, and the like.The use of such media and agents for pharmaceutical active substances iswell known in the art. Except insofar as any conventional media or agentis incompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

By “pharmaceutically acceptable” is meant a material that is notbiologically or otherwise undesirable, i.e., the material may beadministered to an individual along with a CRISP polypeptide withoutcausing any undesirable biological effects or interacting in adeleterious manner with any of the other components of thepharmaceutical composition in which it is contained.

A “subject” or an “individual” is an organism, including, for example, amammal. A mammal may include, for example, a rat, mouse, a primate, adomestic pet, such as, but not limited to, a dog or a cat, livestock,such as, but not limited to, a cow, a horse, and a pig, or a human.Subject also includes model organisms, including, for example, animalmodels, used to study fertilization of an egg by sperm, spermcapacitation, protein tyrosine phosphorylation, or the acrosomereaction.

A “control” sample or subject is one in which a CRISP pathway has notbeen manipulated in any way.

As used herein in vitro is in cell culture, ex vivo is a cell that hasbeen removed from the body of a subject and in vivo is within the bodyof a subject. Unless otherwise specified, “a,” “an,” “the,” and “atleast one” are used interchangeably and mean one or more than one.

The present invention also includes an isolated molecule, the moleculebeing present on the sperm plasma membrane and binds to a CRISPpolypeptide. The molecule may be an isolated component of a calciumchannel or a receptor involved in calcium channel signaling. Forexample, the isolated molecule may be a molecule being present on thesperm plasma membrane and binds to a CRISP-1 polypeptide, or abiologically active analog, fragment, or modification thereof. Thismolecule that binds to a CRISP-1 polypeptide may be an isolatedcomponent of a calcium channel or a receptor involved in calcium channelsignaling.

EXAMPLES

The present invention is illustrated by the following example. It is tobe understood that the particular example, materials, amounts, andprocedures are to be interpreted broadly in accordance with the scopeand spirit of the invention as set forth herein.

Example 1 Role of CRISP Proteins in the Regulation of Sperm Capacitationand Use in Contraception

The results of Example 1 are shown in FIG. 1. Rat sperm were collectedfrom the end of the epididymis and incubated in a defined capacitationmedium in vitro for 5 hours under controlled conditions. A sample ofsperm was taken at the beginning of incubation to provide the time zeroconditions (lane 1 of FIG. 1). Aliquots of collected sperm wereincubated under the following various conditions:

-   -   For 5 hours under non-capacitation conditions (lane 2 of FIG. 1)    -   For 5 hours under capacitating conditions (lane 3 of FIG. 1)    -   For 5 hours under capacitating conditions with increasing        concentrations of CRISP-1 (lanes 4, 5 and 6 of FIG. 1).        At the end of incubation, sperm were solubilized and solubilized        sperm proteins were separated by SDS gel electrophoresis.        Proteins were then transblotted onto a membrane and treated with        a primary anti-phosphotyrosine antibody. The results of this        anti-phosphotyrosine western blot are shown in FIG. 1A. After        recording the results with the anti-phosphotyrosine antibody,        the transblot was striped of antibodies and re-probed with an        antibody against CRISP-1. The results of this anti-CRISP-1        western blot are shown as FIG. 1B.

FIG. 1A shows phosphotyrosine distribution in sperm under the variousincubation conditions. Under non-capacitating conditions, there appearsto be some phosphorylation activity. This phosphorylation activity issignificantly increased under capacitation conditions, with numerousdifferent proteins exhibiting phosphorylation. Phosphorylation activityis inhibited on many proteins by the addition of CRISP-1. FIG. 1B showsthe same gel stained with an anti-CRISP-1 antibody. In lane 1 (timezero) one can see two bands (the D form and the E form of CRISP-1).Under both non-capacitating and capacitating conditions, the D form ofCRISP-1 is lost from sperm (see lanes 2 and 3). When CRISP-1 is addedback (see lanes 4, 5 and 6), the D form re-appears in a dose-dependentfashion. The E form, which is also present in the added CRISP-1 protein,remains constant under all experimental conditions.

Thus, the addition of CRISP-1 to the capacitating incubation mediuminhibits protein tyrosine phosphorylation, a universal indicator ofcapacitation.

Example 2 Inhibition of Capacitation-associated Tyrosine PhophorylationSignaling in Rat Sperm by Epididymal Protein Crisp-1

In mammals, development of fertilizing ability and progressive motilityby sperm, the process of post-testicular maturation, begin as sperm aremoved through the male reproductive tract and are completed when spermare deposited in the female reproductive tract and undergo capacitation.

In the male post-testicular duct system, sperm acquire new proteins andglycoproteins on their surfaces and undergo numerous biochemical changesduring their passage through the ducts that make them capable ofvigorous, directed movement and able to fertilize an egg (Yanagimachi R.Mammalian Fertilization. The Physiology of Reproduction 1994: 186-317).Crisp-1 (DE, AEG) is a glycoprotein couplet (comprised of protein D andprotein E, hereinafter referred to collectively as Crisp-1) that issecreted by the epididymal epithelium (Brooks and Higgins, Journal ofReproduction & Fertility 1980; 59: 363-375; Moore et al, MolecularReproduction & Development 1994; 37: 181-194) and associates with thesperm surface (Rochwerger and Cuasnicu, Molecular Reproduction &Development 1992; 31: 3441; Xu et al, Mol Reprod Dev 1997; 46:377-382.). A portion of the Crisp-1 on the sperm surface, in particularProtein E, is proteolytically-processed (Roberts et al, Biology ofReproduction 2002; 67: 525-533). Crisp-1 is one of manyepididymis-secreted proteins that associate with sperm (Faye et al, BiolReprod 1980, 23: 423-432; Kohane et al, Biol Reprod 1980, 23: 737-742;Moore, J Exp Zool 1981, 215: 77-85 (1981); Wong and Tsang, Biol Reprod1982, 27: 1239-1246; Tezon et al, Biol Reprod 1985, 32: 591-597; Iusemet al, Biol Reprod 1989, 40: 307-316; Vreeburg et al., Bull Assoc Anat(Nancy) 1991, 75: 171-173; Rankin et al., Biology of Reproduction 1992,46: 747-766; Boue et al, Biol Reprod 1996, 54: 1009-1017). Themechanism(s) of the interaction (e.g., covalent bonds, charge effects,hydrophobic bonds) between the sperm plasma membrane and extracellularepididymal molecules is unknown, but is likely to be varied. In contrastseminal vesicle secretions that are known to participate in capacitationin mice (Huang et al., Biol Reprod 2000, 63: 1562-1566 (2000); Huang etal., Biochem J 1999, 343 Pt 1: 241-248; Luo et al, J Biol Chem 2001 276:6913-6921) and bulls (Huang et al, Biol Reprod 2000, 63: 1562-1566(2000); Huang et al, Biochem J 1999, 343 Pt 1: 241-248; Luo et al., JBiol Chem 2001, 276: 6913-6921) are added to the cell surfaces afterejaculation by binding to sperm plasma membrane phospholipid headgroups. In rats there is evidence that seminal vesicle proteins areadded to the sperm surface (Manco and Abrescia, Gamete Res 1988, 21:71-84; Manco et al., Eur J Cell Biol 1988, 47: 270-274), possibly bytransglutaminase activity in semen (Paonessa et al., Science 1984, 226:852-855), and it has been reported also that a prostate-derived proteinbinds to rat spermatozoa (Sansone and Abrescia, J Exp Zool 1991, 259:379-385). Thus, addition of proteins and glycoproteins derived fromdifferent parts of the duct to sperm surfaces occurs throughout the maleexcurrent duct system.

Under normal conditions ejaculated sperm are unable to fertilize an egguntil they have resided in the female tract for a number of hours (thetime varies from species to species (Bedford, Biol Reprod 1970, 2: Suppl2:128-158; Davis, Proc Natl Acad Sci USA 1981, 78: 7560-7564), and haveundergone capacitation. Capacitation was independently, and virtuallysimultaneously, described in two laboratories (Austin, AustralianJournal of Scientific Research, B 1951, 4: 581-589; Chang, Nature 1951,168: 697) as the time required for sperm to penetrate an egg afterhaving been deposited in the female reproductive tract. Residence in thefemale tract is required for capacitation in vivo, resulting in theacquisition of hyperactivated motility in many, but not all species; theloss or changes in some constituents of the plasma membrane, includingproteins and glycoproteins and in the acquisition of the ability toundergo the acrosome reaction.

During the more than half century since its discovery, capacitation hasbeen the subject of intense investigation, particularly since it ispossible to capacitate sperm in vitro and use them to fertilize an egg.Common themes about what happens during capacitation are beginning toemerge. In all species that have been examined, it is necessary forcholesterol to be removed from the membrane, which can be accomplishedin vitro by incubating sperm in a medium containing serum albumin(Davis, Proc Soc Exp Biol Med 1976, 151: 240-243; Davis et al., ProcNatl Acad Sci USA 1980, 77: 1546-1550) or other cholesterol-bindingagents such as cyclodextrins (Choi and Toyoda, Biol Reprod 1998, 59:1328-1333; Visconti et al., Biol Reprod 1999, 61: 76-84). Cholesterolremoval results in a cAMP-dependent tyrosine phosphorylation of a numberof proteins, both in the sperm plasma membrane and in intracellularstructures such as the axoneme and fibrous sheath. Initiation andcompletion of capacitation is absolutely dependent on extracellular Ca⁺⁺and HCO₃ ⁻, in addition to a cholesterol sequestering agent.

In this example we report the results of experiments designed toelucidate the conditions required for in vitro capacitation of ratspermatozoa and the effects of Crisp-1, an epididymal secretory protein,on capacitation. We demonstrate that protein tyrosine phosphorylation, ahallmark of capacitation in other species' sperm, occurs during fivehours of in vitro incubation and that this phosphorylation is dependentupon cAMP. HCO₃ ⁻, Ca⁺⁺, and the removal of cholesterol from themembrane. We also show that Crisp-1, added to the sperm surface in theepididymis in vivo, is lost during capacitation and that addition ofexogenous Crisp-1 to the incubation medium inhibits tyrosinephosphorylation in a dose dependent manner, and thus inhibitscapacitation and ultimately the acrosome reaction. We further show thatthe inhibition of capacitation by Crisp-1 is upstream of the productionof cAMP by the sperm.

Materials and Methods

Chemicals and Reagents: Anti-Phosphotyrosine (4G10) monoclonal IgGconjugated to horseradish peroxidase (HRP) was purchased from UpstateBiotechnology Inc. (Lake Placid, N.Y.). ALEXA FLUOR 488 goat anti-rabbitIgG, AMPLEX Red Cholesterol Assay Kit and Slow-Fade were purchased fromMolecular Probes (Eugene, Oreg.). Cold water fish skin gelatin (40%solution) was purchased from Electron Microscopy Sciences (Washington,Pa.). SUPER SIGNAL West Pico Chemiluminescent Substrate was purchasedfrom Pierce Chemical Co. (Rockford, Ill.). ALBUMAX I lipid-rich bovineserum albumin (BSA) was purchased from Gibco BRL (Grand Island, N.Y.).Original and modified BWW were purchased from Irvine Scientific (SantaAna, Calif.). All other chemicals and reagents were purchased fromSigma-Aldrich (St. Louis, Mo.). Generation of the CAP-A anti-peptidepolyclonal antibody and the 4E9 monoclonal antibody have been previouslydescribed (Moore et al., Molecular Reproduction & Development 1994,37:181-194; Roberts et al., Biology of Reproduction 2002, 67: 525-533).

Media: The base media used for collection and experimental incubation ofsperm was original formula BWW medium (Biggers et al, Methods inMammalian Embryology 1971, 86-116). BWW minus calcium or bicarbonate wasprepared according to the recipe reported by Biggers, et. al. (1971)(Biggers et al, Methods in Mammalian Embryology 1971, 86-116). Spermwere capacitated in BWW with 15 mg/ml ALBUMAX I lipid-rich BSA, unlessotherwise noted. Other cholesterol acceptor molecules included FractionV BSA and methyl-β-cyclodextrin, and were added to BWW in someexperiments.

Sperm Collection and Preparation: Spague-Dawley male retired breederrats were euthanized by CO₂ asphyxiation and epididymes were surgicallyremoved. Radial slits were made in each of the cauda epididymes followedby a 5 minute incubation in 1 ml of BWW buffered with 21 mM HEPES on anorbital shaker to facilitate the swim out of sperm into the media. Thesperm suspensions were placed in a 1.5 ml microcentrifuge tube, leavingbehind the epididymes, and gently shaken by hand to ensure an evenconcentration of sperm. Sperm counts were performed using ahemacytometer. Aliquots of approximately 3.5×10⁶ sperm were diluted into0.5 ml of capacitation medium that was pre-equilibrated overnight at 37°C. in 5% CO₂. The incubation wells were overlayed with 0.5 ml of mineraloil and incubated for times indicated (in figure legends) at 37° C. in5% CO₂. Subjective assessment of sperm motility showed minimal decreasesduring capacitation incubation. All animal experiments were approved bythe Institutional Animal Care and Use Committee of the University ofMinnesota.

SDS-PAGE and Western Blotting: Samples were prepared for SDS-PAGEanalysis using a modification of the protocol described by Visconti et.al., (1995). Briefly, sperm were collected from under oil andcentrifuged at 16,000×g in microcentrifuge tubes for 5 minutesimmediately following the capacitation incubation. The sperm pellet waswashed twice with 1 ml of phosphate buffered saline (PBS) andresuspended in 100 μl of 1× Laemmli sample buffer (Laemmli. Nature 1970,227: 680-685). The samples were vortexed for 15 seconds, heated to 95°C. for 5 minutes and centrifuged at 16,000×g to remove insolublematerial. Supernatants were transferred to new tubes, reduced by theaddition of β-mercaptoethanol (to a final concentration of 2.5%) andheated again to 95° C. for 5 minutes. 20 μl of each sample, equivalentto 7×10⁵ sperm, were subjected to polyacrylamide gel electrophoresis(PAGE) on tris-glycine gels (7.5%, 12% or 15%, depending on theexperiment). Proteins were transferred to Immobilon P membrane(Millipore, Bedford, Mass.) at 100 volts for 1 hour at 4° C.

For detection of tyrosine-phosphorylated proteins, blots were blockedwith 6.5% fish skin gelatin in TBS-T (Tris Buffererd Saline with 0.1%Tween 20) for 30 minutes followed by incubation withanti-phosphotyrosine-HRP antibody (1:15,000), in blocking solution, for1 hour at room temperature. The blots were washed with TBS-T, followedby incubation with HRP substrate (Super Signal West Pico) for 5 minutes.Blots were exposed to X-ray film for 5 to 30 seconds. Western blotdetection of the protein D and E forms of Crisp-1 with anti-peptideantibody CAP-A and monoclonal antibody 4E9 were done as previouslydescribed (Moore et al., Molecular Reproduction & Development 1994, 37:181-194; Roberts et al., Biology of Reproduction 2002, 67: 525-533.).

Immunocytochemistry: Sperm were stained immunocytochemically withanti-peptide antibody CAP-A and monoclonal antibody 4E9 essentially aspreviously described (Moore et al., Molecular Reproduction & Development1994; 37: 181-194). Briefly, sperm were washed 3× in BWW to removemedia, fixed with Bouin's fixative for 30 minutes and washed extensivelywith PBS. Cells were blocked for 30 minutes with 1% BSA/PBS andantibodies were added for an hour incubation at room temperature. Theanti-peptide antibody CAP-A was used at a dilution of 1:200 while mAb4E9 was used at 1:1000. Sperm were washed 3× with PBS and Alexa-Fluor488 anti-rabbit antibody was added to the CAP-A tubes whileanti-mouse—FITC was added to the 4E9 tubes. After incubation for 1 hourin Alexa-Fluor second antibody at room temperature, cells were washedwith PBS mounted on slides in Slow-Fade® and viewed using a Nikonfluorescent microscope.

Cholesterol Assay: Total lipids were extracted from BWW containing MBCDafter incubation with sperm essentially as described by Bligh and Dyer(Bligh and Dyer, Canadian Journal of Biochemistry and Physiology 1959,37: 911-917). Briefly, after incubating sperm with BWW/MBCD, sperm wereremoved by centrifugation and 0.8 ml of supernatant was recovered.Chloroform and methanol were added to the supernatant, with vortexing,to a final ratio of chloroform to methanol to aqueous supernatant of2:2:1.8. After vigorous vortexing, the final mixture was centrifuged for5 minutes at 600×g and one ml of the organic (lower) phase was removedto a new tube. The lipids in the organic phase were dried under a streamof desiccated nitrogen and stored at −20° C.

Cholesterol was measured in the extracted lipid samples using the AmplexRed Cholesterol Assay Kit, according to the manufacturers instructions.Briefly, dried lipid samples were resuspended in 50 μl of reactionbuffer and mixed 1:1 with working solution containing 300 μM Amplex Redreagent, 2 U/ml horseradish peroxidase, 2 U/ml cholesterol oxidase and 2U/ml cholesterol esterase in wells of a 96-well microtiter plate. Astandard curve was prepared using the cholesterol reference standardprovided with the kit. All samples were incubated for 2 hrs at 37° C.Fluorescence of reaction product was measured at various time points ina FL600 Microplate Reader (BIOTEK Instruments, Inc., Winooski, Vt.) withan excitation filter of 530 nm and an emission filter of 590 nm.

Acrosome reaction and staining: The acrosome reaction and assessment ofacrosomal status was performed essentially as described by Bendahmane etal. (Bendahmane et al., Arch Biochem Biophys 2002, 404: 38-47).Following incubation under capacitating or non-capacitating conditionsfor 30 minutes, progesterone (P4), dissolved in DMSO, was added to afinal concentration of 1 μM. After an additional 30 minutes ofincubation, sperm were fixed in 4% paraformaldehyde, washed and dried onslides. To visualize the acrosome, the sperm were stained with 0.22%Coomassie blue G-250 solution for 2 minutes, rinsed with distilled waterand allowed to air dry. Slides were coverslipped using Permount mountingmedia and observed under a Nikon brightfield microscope at amagnification of 600×. For each condition within an experiment, 500cells were assessed for acrosomal status.

Statistical Analysis: All experiments reported in the manuscript wererepeated a minimum of three times. Raw data from the acrosome reactionexperiments were subjected to the Tukey analysis for determination ofstatistically significant differences (P<0.05) between pairs of alltreatment groups.

Results

Initial studies were carried out to characterize the dependence of ratsperm capacitation on the presence of a cholesterol binding molecule,Ca⁺⁺ and HCO₃ ⁻; the three components shown to be requirements ofcapacitation in most other species. Capacitation conditions for ratsperm were tested using tyrosine phosphorylation of sperm proteins as anindication of the extent of the capacitation process. FIG. 2Ademonstrates, by western blot with an antibody against phosphotyrosine,the dependence of capacitation on incubation with a lipid-acceptingmolecule, in this experiment bovine serum albumin (BSA). In the presenceof 15 mg/ml lipid rich BSA, protein tyrosine phosphorylation on spermproteins increased over 5 hours of incubation. Cholesterol wasdetermined to be the lipid responsible for capacitation since incubationwith exogenous cholesterol sulfate inhibited protein tyrosinephosphorylation (FIG. 2B).

Initial capacitation experiments were carried out in solution of a 15mg/ml lipid-rich BSA (Gibco-BRL), a concentration of BSA routinely usedin our BWW solution for in vitro fertilization. Because mostcapacitation experiments are conducted using fraction V BSA, we comparedthe efficacy of using lipid-rich or fraction V BSA at variousconcentrations. FIG. 3A demonstrates that lipid-rich BSA was superior tofraction V for inducing tyrosine phosphorylation in rat sperm at allconcentrations investigated. In fact, incubation of sperm with fractionV BSA gave very low levels of tyrosine phosphorylation in rat sperm.When the same comparison was performed using mouse sperm, where fractionV BSA is routinely used, the efficacy of tyrosine phosphorylation wasthe same (FIG. 3B). These results suggest that different BSApreparations have different effects on sperm depending on the species.The basis for this difference is not clear.

The dependence of rat sperm capacitation on exogenous Ca⁺⁺ is shown inFIG. 4. Incubation in the absence of exogenously added Ca⁺⁺ for 4 hourswas accompanied by minimal tyrosine phosphorylation compared to spermincubated in the presence of 1.7 mM Ca⁺⁺. The level of tyrosinephosphorylation in the absence of exogenous Ca⁺⁺ was higher than thatseen in the absence of BSA, which may be attributable to trace amountsof Ca⁺⁺ in the medium or to the availability of Ca⁺⁺ from intracellularsources. Likewise, capacitation was shown to be dependent on thepresence of bicarbonate ion in the medium by assessing protein tyrosinephosphorylation in the presence and absence of HCO₃ ⁻ (FIG. 5).Solutions in this experiment were buffered with HEPES buffer to insurethat the requirement of bicarbonate was not simply due to its bufferingcapacity in the medium.

To examine the relationship between cholesterol removal from the spermplasma membrane and the protein tyrosine phosphorylation eventsassociated with capacitation, sperm were incubated with two doses of thecholesterol-binding molecule methyl-β-cyclodextran (MBCD). Duringincubation with MBCD, cholesterol was removed from the sperm in adose-dependent fashion. MBCD at 2 mM removed twice as much cholesterolas 1 mM MBCD (FIG. 6A). When protein tyrosine phosphorylation wasmeasured, phosphorylation in 2 mM MBCD was increased in both kineticsand total amount over that observed with 1 mM MBCD (FIG. 6B). Proteintyrosine phosphorylation lagged behind the removal of cholesterol fromthe sperm plasma membrane, as indicated by the fact that cholesterolremoval was at a plateau within 30 minutes with 1 mM MBCD (FIG. 6A) yetno increase in phosphorylation was observed until 2 hours (FIG. 6B).These results indicated that protein tyrosine phosphorylation isdependent on cholesterol removal in a dose dependent fashion, but thatthe kinetics of cholesterol removal is not rate limiting to thephosphorylation process.

The removal of cholesterol from cell membranes has been shown to affectthe organization of lipid micro-domains, or rafts, which in turn canaffect signaling events in the cell (Simons, Nat Rev Mol Cell Biol 2000,1: 31-39). To determine if the removal of cholesterol from ratepididymal sperm might be associated with changes in lipid rafts, spermwere stained with the β subunit of cholera toxin (βCT), which binds tothe ganglioside GM₁ (a lipid known to be present in many lipid rafts)before and after cholesterol removal. Rat epididymal sperm wereincubated in BWW with or without BSA or MBCD to facilitate the removalof cholesterol from the sperm plasma membrane. After 5 hours the spermwere fixed and stained with a fluorescent-tagged β-subunit of choleratoxin, which binds to the sugar moiety of GM₁. In control sperm at timezero or after 5 hours in BWW only, GM₁ staining is tightly confined tothe post-acrosomal and head cap regions of the sperm. After removal ofcholesterol by BSA or MBCD, GM₁ staining begins to diffuse over theequatorial region and acrosome, and increased staining is seen on thesperm tail. Immediately after isolation of sperm from the rat epididymisthe sperm show very specific staining with βCT over the equatorialsegment and the head cap region. This staining pattern remained constantafter 5 hours of incubation in BWW devoid of a cholesterol-bindingmolecule. However, after 5 hours of incubation with 15 mg/ml BSA or 1 mMMBCD, βCT staining became diffuse over the entire sperm head and becamevisible on the sperm tail. Virtually all of the sperm observed (>99%)underwent this redistribution. This result indicates that lipidmicrodomains on sperm are disrupted by removal of cholesterol, and raftcomponents, such as GM₁, are redistributed on the surface of the sperm.This redistribution correlates with sperm capacitation, implicatingraft-associated signaling events in the capacitation process.

Crisp-1 is a sperm maturation protein secreted in two forms, proteins Dand E (Roberts et al., Biology of Reproduction 2002, 67: 525-533., Cameoand Blaquier, Journal of Endocrinology 1976, 69: 47-55; Xu and Hamilton,Mol Reprod Dev 1996, 43: 347-357) by the epididymal epithelium, both ofwhich become bound to the sperm surface during epididymal transit (Mooreet al., Molecular Reproduction & Development 1994, 37: 181-194., Brooksand Tiver, Journal of Reproduction & Fertility 1983, 69: 651-657).Studies have shown that the majority of Crisp-1 is lost from spermduring incubation after ejaculation or after incubation of spermisolated from the epididymis (Tubbs et al., J Androl 2002, 23: 512-521).Staining of the protein D and E forms of Crisp1 by anti-peptide antibodyCAP-A and monoclonal antibody 4E9 in the presence or absence of MBCDreveals that CAP-A binds to both the D and E forms of Crisp-1 andlocalizes to the entire surface of the sperm. With time the staining ofsperm with CAP-A becomes less intense in both the absence and, even moreso, in the presence of MBCD. The intensity of staining with antibody4E9, which recognizes only the E form of Crisp-1, does not change withtime in BWW and decreases only marginally when the sperm are incubatedfor 4 hours with MBCD. Staining, with antibodies that differentiate thebinding of the protein D and E form of Crisp-1, demonstrates that themajority of the protein D and E forms of Crisp-1 is lost duringcapacitation incubation, with or without a cholesterol binding agent.However, the protein E form of Crisp-1 remains confined to the tail ofthe sperm without detectable loss or redistribution during thecapacitation process.

Since the loss of the protein D form of Crisp-1 occurs during the timeframe of sperm capacitation, it is possible that the presence ofexogenous Crisp-1 may inhibit the capacitation process. FIG. 7A showsthe effect on protein tyrosine phosphorylation of incubating sperm undercapacitating conditions in the presence of increasing concentrations ofpurified Crisp-1. At a dose of 400 μg/ml, Crisp-1 inhibits almostcompletely the protein tyrosine phosphorylation associated withcapacitation. Re-probing of these western blots with anti-peptideantibody CAP-A, which recognizes all forms of the Crisp-1 proteins,showed that the endogenous D-form of Crisp-1 (protein D at 32 kDa) islost from the sperm during capacitation and that exogenous protein Dbecomes associated with the sperm coincident with the inhibition ofcapacitation (FIG. 7B). When this western blot was probed with amonoclonal antibody 4E9, which recognizes only the E-form of Crisp-1(protein E at ˜28 kDa), the blot showed that protein E is not lost fromthe sperm surface during capacitation and no additional protein Eassociates with sperm during the incubation with exogenous Crisp-1 (FIG.7C).

It has been recently reported that the protein E form of Crisp-1 isprocessed as it associates with sperm in the epididymis and that aportion of the protein D form of Crisp-1 may also be processed as itassociates with sperm (Roberts et al., Biology of Reproduction 2002, 67:525-533). Comparison of FIGS. 7B and 7C demonstrates the presence of aprocessed form of Crisp-1 that is not recognized by the 4E9 antibody.This observation suggests that the processed forms of Crisp-1 attachpermanently to the sperm while the unprocessed form of protein Dinteracts dynamically with the sperm plasma membrane to reversiblyprevent capacitation-associated tyrosine phosphorylation.

If the tyrosine phosphorylation events suppressed by Crisp-1 representthe suppression of capacitation, then Crisp-1 should also be able toinhibit the ability of the cells to undergo an induced acrosomereaction. To test this, rat sperm were capacitated for one hour with 15mg/ml BSA in the presence or absence of 400 μg/ml Crisp-1 and theacrosome reaction induced with 1 μM progesterone (P4). FIG. 8 shows asignificant increase (P<0.05) in the acrosome reaction in capacitatedsperm after incubation with P4. This increase was completely suppressedby addition of exogenous Crisp-1. The suppression of the acrosomereaction by Crisp-1 was statistically significant (P<0.05). This resultindicates that Crisp-1 is inhibiting capacitation in rat sperm.

The dynamic nature of the interaction between Crisp-1 (unprocessed form)and the sperm surface suggests that the inhibition of protein tyrosinephosphorylation by Crisp-1 may be reversible. To test this possibility,sperm were incubated under capacitating conditions in the presence 200μg/ml Crisp-1 for 5 hours and then removed to capacitation media devoidof Crisp-1. As the data of FIG. 9A demonstrate, significant suppressionof protein tyrosine phosphorylation was observed at 5 hours by Crisp-1.After 3 additional hours of incubation in the absence of Crisp-1,protein tyrosine phosphoryation had resumed and continued out to 24hours. The resumption of phosphorylation activity correlates with theloss of Crisp-1 from the sperm (FIG. 9B).

Previous studies on the requirements for capacitation in mouse spermhave shown that Ca⁺⁺, HCO₃ ⁻, and removal of cholesterol from the spermplasma membrane are all required for the protein tyrosinephosphorylation events of capacitation (Visconti et al., Development1995, 121: 1129-1137). However, the absence of any of these three couldbe compensated for by the addition of cAMP analogs, demonstrating thatcAMP signaling in the sperm is intermediary to protein tyrosinephosphorylation (Visconti et al., Development 1995, 121: 1139-1150).FIG. 10 demonstrates that a similar signaling pathway exists for ratsperm. When sperm were incubated in the presence of the cAMP analogdb-cAMP and the phophodiesterase inhibitor IBMX, protein tyrosinephosphorylation occurred in the absence of any of the three moleculesrequired for capacitation (FIG. 10A). Furthermore, stimulation of thecAMP pathway by db-cAMP and IBMX also overcame the inhibition ofcapacitation caused by exogenous Crisp-1 (FIG. 10B). These resultsindicate that the signaling pathway leading to capacitation is similarbetween mouse and rat, and that Crisp-1 inhibits capacitation byintervening in an event upstream of the stimulation of cAMP productionby the sperm.

Discussion

This study provides the first characterization of the requirements forcapacitation of rat sperm using tyrosine-phosphorylation of spermproteins as the indication that the capacitation signaling cascade hasbeen activated. As with previous work in other laboratories, primarilyusing mouse sperm, we have shown that rat sperm capacitation requiresthe presence of a cholesterol-binding agent, such as BSA, calcium ion,and bicarbonate ion (Visconti et al., Development 1995, 121: 1129-1137;Visconti et al., J Androl 1998, 19: 242-248). Further, the action of allthree of these required molecules likely leads to the production ofcAMP, as evidenced by the ability of exogenous db-cAMP with thephosphodiesterase inhibitor IBMX to overcome the absence of BSA, Ca⁺⁺ orHCO₃ ⁻, consistent with the results of studies of mouse spermcapacitation (Visconti et al., Development 1995, 121: 1129-1137).

Most, if not all, mammalian sperm require cholesterol removal from theplasma membrane in order for capacitation to occur. However, themechanism by which cholesterol removal facilitates capacitation in spermis not known. One likely possibility is that removal of cholesterol fromlipid microdomains, or rafts, facilitates the movement of signalingmolecules in the plasma membrane, allowing critical interactions thatlead to the activation of adenylate cyclase and subsequent tyrosinephosphorylation of target proteins. Several recent studies have providedevidence for the existence of lipid rafts on mouse and guinea pig sperm(Travis et al., Dev Biol 2001, 240: 599-610; Trevino et al., FEBS Lett2001, 509: 119-125.; Honda et al., J Biol Chem 2002, 277: 16976-16984).We demonstrate here that rat sperm contain discrete regions of stainingfor binding of cholera toxin β subunit, which binds to the gangliosideGM₁, a common lipid component of membrane rafts. Furthermore, thediscrete localization of GM₁ is lost during cholesterol extraction witheither BSA or MBCD, suggesting that molecules within the sperm plasmamembrane begin to diffuse upon removal of cholesterol. A similardiffusion of lipids in the sperm plasma membrane has been reported inboar sperm during in vitro capacitation (Gadella et al., J Cell Sci1995, 108 (Pt 3):935-946)).

Our data also show that the degree of tyrosine phosphorylation in ratsperm is dependent upon the extent of cholesterol extraction. The dataof FIG. 6 demonstrate that doubling the amount of MBCD used to extractcholesterol from the sperm membrane increases the maximal degree oftyrosine phosphorylation at the 5 hour time point. Increasing MBCD alsoincreases the kinetics of phosphorylation. Taken together these findingssuggest that, if liberation of signaling molecules to move in the plasmamembrane is the mechanism by which cholesterol extraction works,removing more cholesterol facilitates more interactions and with fasterkinetics. However, it is also clear that removal of cholesterol underthe conditions of our experiments is not rate limiting to subsequenttyrosine phosphorylation. Using 1 mM MBCD, extraction of cholesterolreached a plateau within 30 minutes, but an increase in tyrosinephosphorylation was not detected until 2 hours and is not maximal until3 hours. The delay between cholesterol removal and tyrosinephosphorylation is consistent with a requirement for physicalredistribution of signaling molecules within the plasma membrane.

The requirement for bicarbonate ion in rat sperm capacitation isconsistent with a role for the bicarbonate-dependent soluble adenylatecyclase that has been implicated in the capacitation process in spermfrom other mammalian species (Sinclair et al., Mol Reprod Dev 2000, 56:6-11; Wuttke et al., Jop 2001, 2: 154-158; Flesch et al., J Cell Sci2001, 114: 3543-3555). A previous study using boar sperm demonstratedthat without bicarbonate ion in the media, cholesterol was not lost fromthe plasma membrane during incubation in the presence of BSA (Flesch etal., J Cell Sci 2001, 114: 3543-3555). The authors of this studyproposed that the role of bicarbonate ion was to activate thebicarbonate-dependent adenylate cyclase, which in turn caused thecAMP-dependent activation of flipase, which was required for cholesterolremoval from the plasma membrane. In the work presented here, theabsence of bicarbonate was overcome by addition of cAMP analog and IBMX,consistent with a capacitation requirement for cAMP downstream of therequirement for bicarbonate ion. However, cholesterol removal from themembrane proceeded normally in the absence of bicarbonate ion,supporting a mechanism of capacitation where cAMP acts downstream ofcholesterol removal from the membrane.

Both capacitation and the acrosome reaction are calcium ion dependentfunctions of mammalian sperm (Visconti et al., J Reprod Immunol 2002,53: 133-150); Breitbart, Mol Cell Endocrinol 2002, 187: 139-144). Ourresults demonstrate that exogenous calcium is required for the tyrosinephosphorylation accompanying capacitation, consistent with thisrequirement shown in earlier studies for other mammalian species(Visconti et al., Development 1995, 121: 1129-1137; Dorval et al., BiolReprod 2002, 67: 1538-1545). The specific calcium-dependent molecularevents of capacitation have not been determined, but the ability toovercome the absence of calcium in the medium with exogenous cAMPanalogs suggests that the calcium-dependent events in the sperm areupstream of the activation of adenylate cyclase.

In addition to the requirement for Ca⁺⁺, HCO₃ ⁻, and acholesterol-binding agent in capacitation, a requirement for thedisassociation of Crisp-1 from the sperm membrane for capacitation toproceed in rat sperm has also been demonstrated. It has beendemonstrated by immunocytochemistry that a portion of the Crisp-1staining is lost from the sperm with incubation, primarily from the headregion and by western blot analysis that it is the 32 kDa form ofCrisp-1 that is lost from the sperm membrane (FIG. 7). The addition ofexogenous Crisp-1 inhibits protein tyrosine phosphorylation in areversible manner, suggesting that Crisp-1 interacts with a specificprotein or lipid on the sperm surface, in a dynamic ligand-receptorfashion, and inhibits the capacitation process. Given this effect ofCrisp-1 on rat sperm capacitation and the high concentration of Crisp-1in epididymal fluid, it is likely that Crisp-1 acts as a capacitationinhibiting factor.

Crisp-1 was also shown to inhibit the P4 induced acrosome reaction,supporting the conclusion that Crisp-1 inhibits capacitation and thatprotein tyrosine phosphorylation is required for capacitation in therat. The level of induced acrosome reaction is low compared with thatseen in other species but is consistent with a previous report for ratsperm (Bendahmane et al., Arch Biochem Biophys 2002, 404: 38-47). Thevery high level of spontaneous acrosome reactions that occur in ratsperm with time during capacitation, over 75% by 3 hours, make itdifficult to measure the induced acrosome reaction at extended timepoints where phosphorylation is more easily measured.

The mechanism by which Crisp-1 inhibits the progression of rat sperm tocapacitation is unknown. However, potential mechanisms of action can beinferred from similarities of this protein to proteins of knownfunction. The primary amino acid sequence of Crisp-1 is highly similarto that of many toxins, in particular the toxin helothermine produced bythe lizard Heloderma horridum (Morrissette et al., Biophysical Journal1995, 68: 2280-2288). Helothermine is known to act as an inhibitor ofcalcium flux through the ryanodine receptor, a regulated calcium channelin muscle cells (Morrissette et al., Biophysical Journal 1995, 68:2280-2288). Since calcium is required for capacitation, Crisp-1 mayprevent the uptake of needed calcium via channels in the sperm plasmamembrane. Ryanodine receptors have been reported to be present intesticular germ cells and sperm, but their exact localization remainsunclear (Gianni et al., Journal of Cell Biology 1995, 128: 893-904;Trevino et al., Zygote 1998, 6: 159-172). However, it is certainlyplausible that Crisp-1 acts on the sperm by interacting with a ryanodinereceptor or a ryanodine receptor-like channel in the sperm plasmamembrane.

It appears that Crisp-1 is the only secretory protein of the epididymisto possess capacitation inhibitory activity. However, proteins orfactors in secretions of the male reproductive tract with apparentcapacitation inhibitory activity have been reported from several species(Huang et al., Biol Reprod 2000, 63: 1562-1566 (2000); Aonuma et al.,Chem Pharm Bull (Tokyo) 1976, 24:907-911; Eng and Oliphant, Biol Reprod1978, 19: 1083-1094; Kanwar et al., Fertil Steril 1979, 31: 321-327;Tomes et al., Mol Hum Reprod 1998, 4: 17-25). The mouse seminal vesicleautoantigen has been shown to inhibit protein tyrosine phosphorylationassociated with sperm capacitation and human seminal plasma has beenshown to contain a factor(s) with similar activity (Huang et al., BiolReprod 2000, 63: 1562-1566 (2000); Tomes et al., Mol Hum Reprod 1998, 4:17-25). Although little is known of the mechanism of capacitationsuppression reported in seminal plasma and secretory proteins of theseminal vesicles, it appears that suppression of premature capacitationis an important function of fluids of the male excurrent reproductivetract.

In addition to the 32 kDa form of Crisp-1 that interacts in a reversibleway with the sperm plasma membrane to inhibit capacitation, a secondsmaller molecular weight form is also found on sperm; this form isstrongly attached and is not removed during incubation undercapacitating conditions. It has been previously shown that both the Dand E forms of Crisp-1 are processed (Roberts et al., Biology ofReproduction 2002, 67: 525-533). The processed E form of Crisp-1 isrecognized by monoclonal antibody 4E9 and localizes to the sperm tail;its function there is unknown (Roberts et al., Biology of Reproduction2002, 67: 525-533).

Crisp-1 has been implicated as playing a role in sperm-egg fusion. Anumber of studies in rat, mouse and human systems have shown that fusionof sperm to the plasma membrane of zona pellucida-free eggs is inhibitedin the presence of Crisp-1 (Rochwerger et al., Developmental Biology1992, 153: 83-90); Cohen et al., Biol Reprod 2000, 63: 462-468, Cohen etal., Biol Reprod 2001, 65: 1000-1005). Further, preincubation of zonapellucida-free eggs with Crisp-1, followed by immunocytochemistry withan antibody specific to Crisp-1, demonstrates specific binding sites forCrisp-1 on the surface of eggs (Rochwerger et al., Developmental Biology1992, 153: 83-90). Taken together, these studies suggest that Crisp-1can inhibit sperm-egg fusion and are consistent with the hypothesis thatCrisp-1 is involved in sperm-egg fusion. However, there are no knownfusogenic domains contained within the amino acid sequence of Crisp-1and nothing in the predicted tertiary structure of the protein suggestsa role in membrane fusion. Therefore, it is unlikely that Crisp-1mediates the sperm-egg fusion event directly. Given the ability ofCrisp-1 to block the signaling cascade leading to capacitation, as shownin the present example, a possible role for Crisp-1 in sperm-egg fusionmay involve regulation of signaling events, particularly thoseassociated with protein tyrosine phosphorylation. Processed Crisp-1remaining on the sperm plasma membrane could interact with signalingmolecules on the egg surface to initiate or otherwise regulate thefusion event.

The complete disclosures of all patents, patent applications includingprovisional patent applications, and publications, and electronicallyavailable material (e.g., GenBank amino acid and nucleotide sequencesubmissions) cited herein are incorporated by reference. The foregoingdetailed description and examples have been provided for clarity ofunderstanding only. No unnecessary limitations are to be understoodtherefrom. The invention is not limited to the exact details shown anddescribed; many variations will be apparent to one skilled in the artand are intended to be included within the invention defined by theclaims.

SEQUENCE LISTING FREE TEXT

-   SEQ ID NO: 1-7 are amino acid sequences.-   SEQ ID NO: 8-14 are cDNA sequences.

1. A method of inhibiting sperm capacitation comprising contacting spermthat have not undergone capacitation with a cysteine-rich secretoryprotein (CRISP) polypeptide under capacitation conditions, wherein theCRISP polypeptide has at least 95% sequence identity to human CRISP-1(SEQ ID NO: 1) and the CRISP polypeptide inhibits tyrosinephosphorviation of sperm proteins.
 2. A method of inhibiting spermcapacitation comprising contacting sperm that have not undergonecapacitation with a cysteine-rich secretory protein (CRISP) polypeptideunder capacitating conditions, wherein the CRISP polypeptide is humanCRISP-1 (SEQ ID NO:1).
 3. A method of inhibiting sperm capacitationcomprising contacting sperm with the human CRISP-1 polypeptide SEQ IDNO: 1.